Biochim Biophys Acta. 2015 Feb;1848(2):392-407.

Surface behavior of peptides from E1 GBV-C protein: Interaction with anionic model membranes and importance in HIV-1 FP inhibition.

Galatola R1, Cruz A2, Gómara MJ1, Prat J3, Alsina MA4, Haro I1, Pujol M5.
  • 1Unit of Synthesis and Biomedical Application of Peptides, Department of Biomedical Chemistry, IQAC-CSIC, Jordi Girona 18, 08034, Barcelona, Spain.
  • 2Dept. de Bioquímica y Biología Molecular I, Facultad de Biología, Universidad Complutense, 28040 Madrid, Spain.
  • 3Physical Chemistry Department, Faculty of Pharmacy, University of Barcelona, CSIC-Associated Unit: Peptides and Proteins: Physicochemical Studies, IN2UB Av. Joan XXIII s/n, 08028 Barcelona, Spain; Unit of Synthesis and Biomedical Application of Peptides, Department of Biomedical Chemistry, IQAC-CSIC, Jordi Girona 18, 08034, Barcelona, Spain.
  • 4Physical Chemistry Department, Faculty of Pharmacy, University of Barcelona, CSIC-Associated Unit: Peptides and Proteins: Physicochemical Studies, IN2UB Av. Joan XXIII s/n, 08028 Barcelona, Spain.
  • 5Physical Chemistry Department, Faculty of Pharmacy, University of Barcelona, CSIC-Associated Unit: Peptides and Proteins: Physicochemical Studies, IN2UB Av. Joan XXIII s/n, 08028 Barcelona, Spain. Electronic address: mopujol@ub.edu.

 

Abstract

The interaction between a peptide sequence from GB virus C E1 protein (E1P8) and its structural analogs (E1P8-12), (E1P8-13), and (E1P8-21) with anionic lipid membranes (POPG vesicles and POPG, DPPG or DPPC/DPPG (2:1) monolayers) and their association with HIV-1 fusion peptide (HIV-1 FP) inhibition at the membrane level were studied using biophysical methods. All peptides showed surface activity but leakage experiments in vesicles as well as insertion kinetics in monolayers and lipid/peptide miscibility indicated a low level of interaction: neither E1P8 nor its analogs induced the release of vesicular content and the exclusion pressure values (πe) were clearly lower than the biological membrane pressure (24-30mN m(-1)) and the HIV-1 FP (35mN m(-1)). Miscibility was elucidated in terms of the additivity rule and excess free energy of mixing (G(E)). E1P8, E1P8-12 and E1P8-21 (but not E1P8-13) induced expansion of the POPG monolayer. The mixing process is not thermodynamically favored as the positive G(E) values indicate. To determine how E1 peptides interfere in the action of HIV-1 FP at the membrane level, mixed monolayers of HIV-1 FP/E1 peptides (2:1) and POPG were obtained. E1P8 and its derivative E1P8-21 showed the greatest HIV-1 FP inhibition. The LC-LE phase lipid behavior was morphologically examined via fluorescence microscopy (FM) and atomic force microscopy (AFM). Images revealed that the E1 peptides modify HIV-1 FP-lipid interaction. This fact may be attributed to a peptide/peptide interaction as indicated by AFM results. Finally, hemolysis assay demonstrated that E1 peptides inhibit HIV-1 FP activity.

KEYWORDS: AFM; Fluorescence technique; GBV-C peptide; HIV-1 FP; Hemolysis; Lipid model membrane

PMID: 25450346

 

Supplementary material      

Interaction E1P8 derivative peptides with HIV-1 FP: thermodynamic parameters

The mean thermodynamic parameters related with peptide-peptide interaction were obtained by means of miscibility studies of E1P8 and its analogues with HIV-FP.

The capacity of each peptide to form monolayers was performed by the compression isotherms of pure parent peptide and its analogues. A transition can be seen, around surface pressure 35 mN/m, for E1P8 peptide monolayer, which is observed as a pseudoplateau in the course of the compression isotherm, as it has shown for other peptides with similar characteristic (Sanchez-Martin, M.J., 2010). For E1P8-12 monolayer was detected about 25 mN/m, while E1P8-13 and E1P8-21 monolayers result in a continuous rise of isotherm. This plateau could be the result of the formation of a bilayer (Malcolm, B.R. 1971; Malcom, B.R., 1985), or of molecular segments being partially lifted from water surface (Kaku, M. et al., 1992), or of a change in orientation of the molecules upon compression (Larios, C., 2006; Hac-Wydro, K. 2007). The compression isotherms of each of them and their mixtures at different molar concentration was carried out and the surface compression modulus for all systems was calculated according to the Eq. (1) where A is the area per molecule at the indicated surface pressure and π is the corresponding surface pressure.

e1   Eq. (1)

MPC fig1

Fig. 1. Surface pressure-mean molecular area (π-A) compression isotherms of mixtures of HIV-1 FP and E1P8, E1P8-12, E1P8-13 and E1P8-21 spread on a HEPES subphase (pH 7.4). Inset: Plots of compression modulus (Cs-1) as a function of the surface pressure.

 

The changes of isotherms shape when HIV-1 FP was mixed with E1P8, E1P8-12, E1P8-13 and E1P8-21 are shown I Fig.1. In all cases clearly a peptide-peptide interaction is seen. In the mixtures with E1P8 and E1P8-12 we can observe a pseudoplateau that decreases as HIV-1 FP mole fraction increases while when HIV-1 FP is added to E1P8-13 and E1P8-21 solution produces an expansion of monolayer. The surface compression modulus can be used to characterize the phase state of the monolayer (for liquid expanded films, compressibility ranges from 12.5 to 50 mN/m, while, for the liquid condensed phase, it ranges from 100 to 250 mN/m) (Alsina, M.A., 2006, Davies,J.T., 1961, Maget-Dana, R., 2001). The surface compressibility modulus (Fig. 1 inset) indicates that the HIV-1 FP monolayer is in liquid expanded until it collapses. The addition of peptides, except E1P8-21, causes an increase of surface compressibility modulus, but the values of C-1s remain less of 50 mN/m, indicating that the mixtures monolayers are in liquid expanded during the compression.

 

MPC fig2

Fig. 2. Plots of Area for mixtures of E1P8, E1P8-12, E1P8-13 and E1P8-21 with FP as function of the FP mole fractions for mixed monolayers.

 

The peptide-peptide miscibility were evaluated by the application of the additivity rule. The additivity rule predicts that in a two-component system, if the area per molecule at different mole fractions of each component is a linear combination of the values of the area per molecule for each component multiplied by its mole fraction. This is the case when there is either totally ideal mixing or complete immiscibility of the components. In the case that the area per molecule shows deviation from ideality, it suggests that the components are miscible and they interact by means repulsive interactions if the deviations are positive, while there are attractive interactions if the deviations are negative (Chattoraj, D.K., et., 1991). The plots of mean molecular area (that of the contribution of both peptides) as function of the HIV-1 FP ratio at a given and constant surface pressure (Fig.2) reveal in general a linear combination of the values of the area per molecule and its mole fraction. In general, small positive deviations were observed in the mixtures of peptides with HIV-1 at every pressure, indicating repulsive interaction between the components, except E1P8-12/FP, at 5 mN/m surface pressure, where small negative deviations were shown, indicating attractive interaction between peptide.

To better qualify the miscibility of E1P8, E1P8-12, E1P8-13, E1P8-21 with FP, the excess free energy of mixing (ΔGMEX) associated to this process have been calculated, using equation described in Pagano et al., 1972 Eq. (2), where NA is Avogadro’s number, A12 is the mean area per molecule in the mixed film, A1 and A2 are the areas per molecule in the pure films, X1 and X2 are the molar fractions, and π is the surface pressure (mN/m).
e2

Eq. (2)

 

Table 1. The excess free energy of mixing (ΔGMEX) (J/mol) in mixed monolayers of E1P8, E1P8-12, E1P8-13, E1P8-21 and FP.

 MPC tab1

 

Table 1 shows the excess free energy of mixing (ΔGMEX) values for peptide/peptide mixtures. AS can be seen just the mixtures E1P8-12/FP at 25 mN/m of surface pressure present the |ΔGMEX| values lightly higher than the product of RT (2477.5 J/mol, at 24 °C) while everything else mixtures can be considered ideal (Gaines, G.L.,1966), due the same hydrophobic profile based on their primary sequence of FP, E1P8 and its analogues. These results are in agreement with the conclusions obtained in our previous work. These results reinforce our previous work. The replacement of residues not modify significantly the thermodynamic behavior of mixtures of HIV-1 FP and GBV-C peptides assayed: E1P8 and derivatives.

 

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